s2fieldsofstudy: Biology Medicine

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Antitumor Effect of Traditional Drugs for Neurological Disorders: Preliminary Studies in Neural Tumor Cell Lines

Glioblastoma multiforme is the most common malignant primary brain tumor in adults. Despite new treatments developed including immunomodulation using vaccines and cell therapies, mortality remains high due to the resistance mechanisms presented by these tumor cells and the function of the blood–brain barrier that prevents the entry of most drugs. In this context of searching for new glioblastoma therapies, the study of the existing drugs to treat neurological disorder is gaining great relevance. The aim of this study was to determine, through a preliminary in vitro study on human glioblastoma (A172, LN229), anaplastic glioma (SF268) and neuroblastoma (SK-N-SH) cell lines, the possible antitumor activity of the active principles of several drugs (levomepromazine, haloperidol, lacosamide, valproic acid, levetiracetam, glatiramer acetate, fingolimod, biperiden and dextromethorphan) with the ability to cross the blood–brain barrier and that are commonly used in neurological disorders. Results showed that levetiracetam, valproic acid, and haloperidol were able to induce a relevant synergistic antitumor effect when associated with the chemotherapy currently used in clinic (temozolomide). Regarding the mechanism of action, haloperidol, valproic acid and levomepromazine caused cell death by apoptosis, while biperiden and dextromethorphan induced autophagy. Fingolimod appeared to have anoikis-related cell death. Thus, the assayed drugs which are able to cross the blood–brain barrier could represent a possibility to improve the treatment of neural tumors, though future in vivo studies and clinical trials will be necessary to validate it.

Introduction
Among neural tumors, glioblastoma or glioblastoma multiforme is the most common malignant primary brain tumor in adults (45.2%) (Kanderi and Gupta 2022) with a worldwide incidence between 0.59 and 5 cases per 100,000 and a progressive increase due to the aging population among other factors. The median age at diagnosis is 62 years and the average survival is approximately 14.6 months (Grech et al. 2020) being one of the most aggressive tumors. Usually, their location is supratentorial (frontal, temporal, parietal and occipital lobes) and it is rarely located in the cerebellum. Currently, tumor surgical resection with maximum safety followed by radiotherapy and concurrent chemotherapy with Temozolamide is the main treatment (Kanderi and Gupta 2022). Other novel treatments include boron neutron capture therapy (BNCT), anti-angiogenic therapy, gene and epigenetic therapy, and the use of oncolytic viruses (Zhang et al. 2019). In addition, new therapeutic strategies including immunomodulation for glioblastoma using vaccines, cell-based therapies, and therapies that regulate immune checkpoints are being developed (Reardon et al. 2020;Wen et al. 2020;Medikonda et al. 2021).
In this context of searching for new glioblastoma therapies, strategies based on the existing drugs to treat neurological disorder have been analyzed. In fact, antipsychotics have shown promising results, mainly because they are able to cross the blood-brain barrier (Kamarudin and Parhar 2019). In addition, Lee et al. (2016) expose that anticonvulsivants such as oxacarbazepine or valproic acid show antitumor activity against glioblastoma cell lines and enhances temozolomide cytotoxicity (Lee et al. 2016). Moreover, Yamada et al. (2020) demonstrated the antitumor effect of riluzole, a glutamate receptor inhibitor, against glioblastoma cell lines by MGMT suppression and temozolomide sensibilization (Yamada et al. 2020).
Finally, in the last 30 years, the survival rates of glioblastoma patients have shown no improvement. The future of its treatment will depend on clinical and genomic studies that allow us to understand the origin and development of this complex disease. Meanwhile, the search for new treatments that improve the prognosis of these patients is necessary. The aim of this manuscript was to determine, through a preliminary in vitro study, the possible antitumor activity of the active principles of several drugs with the ability to cross the blood-brain barrier and that are commonly used in neurological disorders.

Cell Viability Assay
To investigate the effect of active principles on human glioblastoma cell proliferation, SF-268, A-172, SK-NSH (5 × 10 3 cells/well) and LN-226 (8 × 10 3 cells/well) were seeded in 48-well plates and incubated overnight. After 24 h, cell cultures were exposed to increasing concentrations of active principles (72 h) determining cell viability. Cells were fixed with 10% trichlooacetic acid (TCA) (20 min at 4 °C). Once dried, the plates were stained with 0.4% sulforhodamine B (SRB) in 1% acetic acid (20 min, in agitation). After three washes with 1% acetic acid, SRB was solubilized with Trizma ® (10 nM, pH 10.5). Finally, the optical density (OD) was measured at 492 nm in a spectrophotometer EX-Thermo Multiskan. Cell survival (%) was calculated according to the following equation: Cell survival (%) = Treated cells OD -blank/Control OD − blank × 100. In addition, half maximal Inhibitory Concentration (IC50) was calculated (GraphPad Prism 6 Software, La Jolla, CA, USA). For the combination effect, the combination index (CI) was calculated using the Compusyn software (Chou and Martin 2005), where a CI > 1 indicates antagonism, where a CI level of < 1 indicates synergy and a CI level equal to 1 indicates additivity. IC30 doses of the studied compounds and IC25 doses of temozolomide were used for combination studies.

Wound-Healing Assay
To determine the tumor cell migration capacity of cell lines and, therefore, their invasiveness and ability to generate metastases, an in vitro migration assay was performed with SF-268 cell line. Cells were seeded in 12-well plates with 3 × 10 5 cell/well in 1 mL of culture medium and grown to 100% confluence in standard culture conditions. Once confluence was reached, a "wound" was made with a 100 µL sterile pipette tip followed by washing with PBS to remove the detached cells and the medium was substituted for serum-free DMEM. Immediately, cells were exposed to the active principles (non-cytotoxic dose, IC5-IC15) for 72 h. Images were taken with an inverted light microscope Olympus CKX41 (Olympus Corporation) at different times (0, 8, 24, 48, and 72 h) to observe cell migration in comparison to the control (cells without treatment). To evaluate the effect of the active principles, the percentage of migration was calculated by measuring the area free of tumor cells at different times (Image J software).

Tunel Assay
To detect the apoptosis based on the detection of single-and double-stranded DNA breaks a tunel assay was carried out. SF-268 (3 × 10 4 cells) were seeded in 8 well culture slides (Corning, USA), exposed to active principles (IC 50 ) for 48 h, fixed with 100 µL of paraformaldehyde (4% in PBS, pH 7.4) and incubated 60 min at 25 °C. Then, cells were washed with PBS and resuspended with 100 µL/well permeabilisation solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice at 4 °C. Cells were washed again with PBS, resuspended in 50 µL/wll Tunel reaction mixture (Sigma Aldrich, Madrid, Spain) and incubated 1 h at 37 °C in a humidified atmosphere in the dark. Finally, cells were washed with PBS and observed by fluorescence microscopy (Leica Microsystems, Wetzlar, Germany).

Lysotracker Labeling
To determine the apoptosis by autophagy, SF-268 (3 × 10 4 cells) were seeded in 8 well culture slides (Corning, USA), exposed to active principles (IC 50 ) for 24 h and stained and loaded with LysoTracker ® Red DND-99 (Thermo Fisher Scientific, Waltham, MA, USA) 50 nM for 30 min at 37 °C, a fluorescent red dye used for labeling and monitoring of acidic organelles. Cells were washed again with PBS and stained with Hoechst (1:1000). Finally, cells were observed under fluorescence microscopy (Leica Microsystems, Wetzlar, Germany).

Statistical Analysis
Statistical analysis was performed by IBM SPSS Statistics 26.0 and GraphPad Prism 8. All the data were presented as the mean value with standard deviation (SD). All experiments were performed in triplicate. After the homogeneity test of variance, t-test was performed to compare the differences between groups with equal variance, while F-test was used for groups with uneven variance. Significance values were denoted by (*) p < 0.05 significant; (**) p ≤ 0.01 highly significant.

Antiproliferative Activity in Cultured Cells
As shown in Table 1, the active principles analyzed showed very different antitumor effects on human glioblastoma lines although in all cases this effect was dose-dependent. Thus, levomepromazine was the drug that showed the lowest

Active Principles Enhanced the Temozolomide Antiproliferative Effect in SF-268 Cells
As shown in Fig. 1, the active principles of some drugs combined with TMZ induced synergistic effects compared to monotherapies (CI < 1). The greatest synergistic effect was dependent not only on the used drug but also on the cell line analyzed. In fact, dextromethorphan, haloperidol, and levetiracetam in combination with TMZ showed the highest antiproliferative activity and synergistic effect on the A-172 cell line. Similarly, haloperidol and lacosamide + TMZ showed the greatest effect on the LN-229 cell line. Regarding the SF-268 cell line, haloperidol and levetiracetam associated with TMZ showed the highest synergistic activity. Finally, in the SK-N-SH cell line, dextromethorphan + TMZ showed the highest antiproliferative and synergistic effect. Valproic acid, haloperidol and leveraracetam showed a synergistic effect with TMZ in all studied cell lines.

Analysis of Cell Migration
To assess the effects of the glatiramer acetate and fingolimod on tumor cell migration capacity, the migration of SF-268 cultured cells were analyzed by a cell wound healing assay. Results showed that non-cytotoxic doses of both drugs induced a decrease in tumor cell migration vs. control (nontreated) cells. In fact, the modulation of migration could be detected as early at 24 h after wound induction. However, the most significant reduction of cell migration was detected at 72 h (p < 0.05) (Fig. 2).

Molecular Analysis of Cells Death Induction by Active Principles
Tunel assay was conducted to determine apoptosis mechanisms of the active principles. As shown in Fig. 3, haloperidol, levomepromazine and valproic were the drugs that induced a larger population of in SF-268 cells entering apoptosis (p < 0.05). On the other hand, Lysotracker assays determined that biperiden and dextromethorphan were the only drugs that induce a significant formation of autophagic vesicles in these same cells (p < 0.001).

Discussion
Despite improvements in neurosurgical techniques, radiotherapy, development of new chemotherapeutics agents and the comprehensive genomic and molecular characterization, GBM remains one of the most aggressive brain tumors. In recent years, the possible antitumor effect exerted by some drugs used for neurological disorders that could help the treatment of GBM is gaining interest. Our results showed that levomepromazine and fingolimod were the drugs with a higher antitumor effect reaching an IC50 between 16 and 50 µM in the first case and 16 µM and 23 in the second case, in neural tumor cell lines tested. In the case of levomepromazine, the antitumor activity could be explained by its binding and inhibition of translationally controlled tumor protein (TCTP), which is a novel target for differentiation therapy because it is down-regulated in tumor reversion experiments (Seo and Efferth 2016;Tripathi et al. 2021). On the other hand, the fingolimod effect may Fig. 1 Combination indexes of different compounds with TMZ in different neural tumors cell lines. Combination index (CI) was calculated using the Compusyn software, where a CI > 1 indicates antagonism, where a CI level of < 1 indicates synergy and a CI level equal to 1 indicates additivity. IC30 doses of the studied compounds and IC25 doses of temozolomide were used for combination studies be related to the inhibition of multifunctional sphingosine-1-phosphate (S1P), a molecule involved oncogenesis, proliferation, migration and metastasis and that is overexpressed in GBM. In fact, this target has been described as a new therapeutic strategy in GBM (Mahajan-Thakur et al. 2017). In addition, the fingolimod cell death mechanism could be related to anoikis as suggested in our results showing detached and rounded cells after treatment.
Interestingly, some active principles not only exerted an antitumor effect against neural tumor cells but also showed a synergistic effect when associated with TMZ. Such is the case of haloperidol, levetiracetam and valproic in all studied cell lines, and, dextromethorphan in the A-172 and SK-N-SH cell lines or biperiden in SF268 and SK-N-SH cell lines. In our results, biperiden and dextromethorphan mainly induced autophagic cell death and valproic, haloperidol and levomepromazine, apoptotic cell death. It is known that haloperidol was able to induce apoptosis and increase caspase-8 expression in other glioblastoma cell lines (Papadopoulos et al. 2020). On the other hand, dextromethorphan could exert its antitumor effect through its activity as an agonist of ơ1 receptors that are overexpressed in many tumors of neural and non-neural origin (Mégalizzi et al. 2007). In addition, previous studies showed that levetiracetam inhibited tumor cell growth by decreasing the expression of O6-methylguanine-DNA methyltransferase (MGMT). This MGMT modulation may increase the p53 protein binding to the promoter region of the gene coding for that enzyme, through recruitment of the mSin3A/histone deacetylase 1 (HDAC1) complex. Thus, our results suggest that levetiracetam could increase the GBM sensitivity to temozolomide, with a consequent potential therapeutic benefit (Roh et al. 2020). In fact, Pallud et al. (2022) published an observational study in which the intake of oral levetiracetam during glioblastoma chemoradiation with temozolomide increased significantly overall survival in those patients (Pallud et al. 2022).
Finally, our study also highlights the ability of glatiramer acetate and fingolimod to inhibit GBM cell migration. This effect is probably related to their activity on cell adhesion receptors to the extracellular matrix (Mathias et al. 2017). Glatiramer acetate is a myelin receptor inhibitor and fingolimod is a sphingosine receptor blocker. In addition, biperiden produced, mainly, autophagy in glioblastoma cells, which could be explained by blocking MALT1 protease activity, which increases autophagy and culminates in lysosome-mediated cell death and concomitantly with the inactivation of mTOR (Jacobs et al. 2020).

Conclusion
In conclusion, our results demonstrated that some traditional neurological compounds, such as neuropleptics, antiepileptics or immunomodulators, among others, showed significant antitumor activity against neural tumor cell lines in vitro. Furthermore, some of them (especially levetiracetam, valproic acid, and haloperidol) were able to induce a relevant synergistic antitumor effect when associated with TMZ. Regarding the mechanism of action, haloperidol, valproic acid and levomepromazine caused cell death by apoptosis, while biperiden and dextromethorphan induced autophagy. Fingolimod appears to have anoikis-related cell death. Thus, the assayed drugs which are able cross the blood-brain barrier, represents a possibility to improve the treatment of neural tumors. Future in vivo studies and clinical trials will be necessary to validate this new use. Funding Funding for open access charge: Universidad de Granada / CBUA. This work was supported by the Project Innbio INB-009 (Granada University and ibs. GRANADA) and by the CTS-107 Group of the Junta de Andalucía (Spain).

Availability of data and materials
The datasets used can be accessed on request from the corresponding author.

Competing interests
The authors declare no competing interests.

Disclaimers
The authors indicate that the views expressed in the submitted article are their own and not an official position of the institution or the funding agency.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Fig. 3 Mechanism of cells death by active principles. A TUNEL assay in SF268 cell line with different drugs. Fluorescence images and intensity graphics. (*) p < 0.05 significant; (**) p ≤ 0.01 highly significant, (***), p ≤ 0.001 very highly significant. B Lysotracker assays in SF268 cell line with different drugs. Fluorescence images and intensity graphics. (*) p < 0.05 significant; (**) p ≤ 0.01 highly significant, (***), p ≤ 0.001 very highly significant ◂


Natural Spider Silk Nanofibrils Produced by Assembling Molecules or Disassembling Fibers

Spider silk is biocompatible, biodegradable, and rivals some of the best synthetic materials in terms of strength and toughness. Despite extensive research, comprehensive experimental evidence of the formation and morphology of its internal structure is still limited and controversially discussed. Here, we report the complete mechanical decomposition of natural silk fibers from the golden silk orb-weaver Trichonephila clavipes into ~10 nm-diameter nanofibrils, the material's apparent fundamental building blocks. Furthermore, we produced nanofibrils of virtually identical morphology by triggering an intrinsic self-assembly mechanism of the silk proteins. Independent physico-chemical fibrillation triggers were revealed, enabling fiber assembly from stored precursors"at-will". This knowledge furthers the understanding of this exceptional material's fundamentals, and ultimately, leads toward the realization of silk-based high-performance materials.

Introduction
Spider silk features an impressive tensile strength and toughness, outperforming many man-made engineering materials [1][2][3][4][5]. This high-performance protein-based material is fully sustainable and produced under ambient temperature and pressure, using a small amount of energy [4]. Furthermore, its biocompatibility makes it attractive for biomedical applications.
Because of these outstanding traits and prospects, spider silk has been intensely studied. Yet, details of the structure and formation process of the impressive high-performance silk fibers are still not understood well enough [6][7][8] to enable the synthesis of silk-inspired fibers with comparable properties [7,[9][10][11]. Here, we demonstrate that the native silk protein has a pronounced preference to form nanofibrils, about 10 nm in diameter: we have found these nanofibrils to represent the bulk of naturally produced spider silk fibers, and we have found that they can be triggered to naturally self-assemble from native silk dope in vitro. This has wideranging implications for the understanding of the structure of spider silk fibers, their structureproperty relationships, and their synthesis.
Nanofibrils have been shown to be the basic building blocks of many strong and hard biological materials such as exoskeletons of crustaceans (chitin), wood (cellulose), tissues/tendons/cartilage (collagen), as well as the silk of silkworms [12,13]. In contrast, the role and prevalence of nanofibrils within spider silk, a material providing a unique combination of high strength and high extensibility, has not yet been fully established. While several widely used models suggest silk nanofibrils as the predominant structure in the core of the fibers, there is hardly any experimental evidence demonstrating that they are indeed present in spider silk fibers in large amounts [1,2,6,[14][15][16]. As a matter of fact, some studies still promote globular proteins as basic structural elements of spider silk [17,18]. Moreover, studies detecting nanofibrils in spider silk feature widespread regarding their lengths and diameters between different species or even within a single species [6,[19][20][21][22]. We recently discovered that the tapelike silk of the Chilean recluse spider [23] is solely made out of 20 nm-diameter nanofibrils, which are oriented strictly parallel to the direction of the fiber [24]. While this observation provides important support for the idea that nanofibrils are pivotal for the structure and performance of spider silk, it is not clear whether the results of this relatively uncommon spider silk readily apply to most or all spider silks. The most commonly studied species are orb weavers featuring cylindrical fiber morphologies, in particular the Trichonephila, Araneus, or Argiope genera, which have become key model systems [3].
The exfoliation of silk fibers, predominantly from silkworms, has recently been studied and demonstrated by several groups [25][26][27][28][29][30][31]. This technique offers the prospect of reshaping silk into other form factors without losing some of its key traits, and it is thus interesting for many applications, such as making papers [30,31], filter membranes [27], sensors, and electronic devices [25,26]. In addition, these techniques can also provide an insight into the internal structural makeup of the fiber. Achieving a high degree of exfoliation without excessive protein degradation is challenging; chemical and mechanical treatments and combinations thereof have been used. The chemical treatments have included acid hydrolysis [32], high concentrations of urea [33], deep eutectic solvents (guanidine hydrochloride and urea) [27], hexafluoroisopropanol (HFIP) [34], and NaOH with urea [35]. As mechanical means, ultrasonication and high-shear mixing have been employed [28,30,36]. Most of these efforts have yielded nano-or microfibrillar materials; however, depending on the experimental techniques used, a widespread in terms of the degree of exfoliation, fibril diameters, and diameter distributions have been reported, ranging from 3 nm to hundreds of nanometers [6,12]. For spider silk, on the other hand, only two studies have attempted exfoliation, without providing information on the exfoliation yield or the role of nanofibrils within the silk fiber [36,37]. In particular, exfoliation has not been reported for the important silk of orb-weaving spiders.
Studying exfoliation can reveal the prevalence and properties of nanofibrils within a silk fiber, which is important knowledge for the design of synthetic, silk-inspired fibers. However, for the synthesis of such fibers, it is equally important to know how these fibrils come into existence, starting from the aqueous silk dope, and how they become oriented within the fiber. Interestingly, we have been able to demonstrate that native silkworm silk proteins (fibroins) taken directly from the gland can be triggered to self-assemble into long, straight ≈20 nm nanofibrils closely resembling the nanofibrils observed in natural or exfoliated silkworm silk by applying shear [38]. This capability was absent in reconstituted silk proteins [39]. Similar types of self-assembly have also been studied with recombinant spider silk proteins (spidroins), even to synthesize macroscopic fibers [7,[40][41][42]. However, these recombinant spidroins currently do not implement the full sequence of their natural counterparts, and the materials made from these recombinant spidroins typically exhibit lower performance than natural spider silk [7][8][9]40].
Recently, the early, pre-fibrillar stages of self-assembly from hierarchical micellar subdomains induced by shear have been studied in native Latrodectus hesperus spidroin by nuclear magnetic resonance and cryo-transmission electron microscopy [43]. However, the self-assembly of native spidroin nanofibrils has not been reported yet.
To produce protein fibers that can be spontaneously produced from a protein reservoir as in the natural system, one needs not only an effective self-assembly mechanism to produce nanofibrils with high yield, but also a way to prevent premature assembly of the stored protein.
To prevent premature assembly inside the storage sack, silk protein is stored in an environment with neutral pH and high Na + and Cl − ion concentrations [1,[44][45][46][47][48][49]. It has been proposed that in these conditions, spidroins form micellar-like structures to prevent aggregation and unwanted fibrillation. When the silk dope travels through the S-shaped tapering duct, the pH reduces, and Na + and Cl − ions exchange with K + and ions [1,[44][45][46][47][48][49]. These chemical changes with the help of physical shearing force generated by the tapering duct trigger the self-assembly of aligned nanofibrils from the silk dope. Here we report the preparation of spider silk nanofibrils using both disassembly of native fibers via exfoliation and self-assembly of molecules from native silk dope (Fig. 1). In both cases, the silk was sourced from Trichonephila clavipes (Fig. 1A), arguably the most well-studied system for mechanically high-performing silks. For exfoliation, we collected major ampullate (MA) silk naturally spun by spiders living in large cages (Fig. 1B). Exfoliation was done using an ultrasonic homogenizer (Fig. 1C) until the macroscopic silk fibers disintegrated (Fig. 1D). For self-assembly, we dissected live spiders to extract the major ampullate silk gland (Fig. 1E) and subsequently diluted it using deionized water to prepare solutions with a range of concentrations.
These solutions were spin-coated onto dry mica substrates, a process that shears and concentrates the silk solutions within seconds, not unlike what happens in the spider's spinning duct during the natural spinning process (Fig. 1F). Interestingly, dynamic-mode atomic force microscopy (AFM) scans of these samples revealed that both of these approachesexfoliation and selfassemblyyielded uniform nanofibrils featuring similar morphologies and similar diameters of ≈10 nm (Fig. 1G). Our findings suggest that the formation of spider silk nanofibrils from an aqueous solution of spidroin is a robust process and an intrinsic capability of these proteins.
Notably, we also observed that even in the presence of shear force, this self-assembly process could be prevented by maintaining a solution chemistry similar to what is found in the storage sack: a close to neutral pH and a high concentration of Na + and Cl − ions. These findings show for the first time that physical and chemical conditions are both critical controlling factors for the nanofibril formation from native spidroin. This insight into the spinning conditions is particularly important because nanofibrils have been shown to play a decisive role in the outstanding structural properties of silk by experimental and theoretical studies. Thus, our work provides significant inspiration for ongoing efforts to synthesize artificial yet sustainable silk-like fibers with strength and toughness comparable to the natural material.

Sample Preparation
Mature female Trichonephila clavipes spiders were captured in the wild in Citrus County, Florida. They were kept in a cage made of Plexiglass strips and aluminum mesh and were fed one cricket per week.
For exfoliation experiments, naturally spun silk was collected from the cages of healthylooking spiders; silk (3.4 mg) was added to deionized water (60 mL) (Millipore Synergy UV).
Ultrasonication (750 W, 20 kHz, 40% amplitude) was applied to the solution (Cole-Parmer CPX750 Ultrasonic Homogenizer) at a controlled temperature of 50 °C. Ultrasonic pulses of 10 seconds duration followed by a 10-second pause were run for 30-135 minutes to obtain the desired level of exfoliation. Then, 40 μL of the resulting solution was sampled and spin-casted (Laurell WS-400Bz-6NPP) onto freshly cleaved mica sheets (2 mins at 2000 rpm) for imaging.
For self-assembly experiments, the major ampullate glands were obtained as described by Jeffery et al. [50] Then, forceps were utilized to poke a hole into the glycoprotein layer of the gland to expose the silk dope inside. The dope was then placed in a microcentrifuge tube containing water (1 mL) (or PBS buffer for the experiments we did to analyze the effect of ions) and left to homogenize overnight in a refrigerator to prevent the proteins in the dope from denaturing. To determine the original concentration of the stock solution, a drop of known volume V s from the solution was placed on a substrate of mass m s , which was then placed in a vacuum oven at 60 °C to dry overnight, and the mass m l of the dried sample was measured. The concentration c of the stock solution was then calculated as c = (m lm s )/V s . Dilutions of 1000 mg/L, 100 mg/L, and 10 mg/L were then prepared from these stock solutions. 40 μL of solution were micro-pipetted onto freshly cleaved mica and spin-coated (Laurell WS-400Bz-6NPP) for 2 minutes at 2000 rpm.

Atomic Force Microscopy
NTEGRA Prima Scanning Probe Laboratory (NT-MDT, Zelenograd, Russia) was utilized to scan both exfoliated and self-assembled samples at room temperature. To minimize the deformation caused by tip-sample interactions, dynamic mode imaging was conducted using μmasch HQ: NSC15/Al BS AFM tips featuring a radius of curvature r≈8 nm, a resonance frequency f≈325 kHz, and a spring constant k≈40 Nm −1 . Gwyddion software was used to flatten and analyze the AFM topography images (http://gwyddion.net/). Blender software (https://www.blender.org/) was used to prepare 3D topography images and 3D rendered illustrations of the proposed silk structure.

Scanning Electron Microscopy
A Hitachi S-4700 field emission electron microscope (FESEM) equipped with a secondary electron detector was used to image disintegrated silk fibers. The samples were run at an acceleration voltage of 10 kV with a 7.5-8.0 mm working distance. For sample preparation, disintegrated silk fibers were dried on a mica sheet and attached to an aluminum SEM sample holder via conductive tape. The sample was sputter-coated (Anatech LTD, Hummer 6.2) with ≈2 nm layer of gold/palladium prior to FESEM imaging to prevent charging.

Statistical Analysis
The diameter measurements of fibers in SEM images were done using the ImageJ image analysis software (https://imagej.nih.gov/ij/). The diameters of nanofibrils in AFM images were measured using the following procedure. Topography sections perpendicular to nanofibrils were first produced from AFM topography data using the Gwyddion AFM image processing software.
The topography profiles of the fibrils were then fitted with Gaussian functions, and the full width at half-maximum values of these fitted curves were taken as the diameters of nanofibrils. This criterion was used to compensate for the broadening induced by the size of the AFM probe, which is comparable to the fibril diameters. All data analysis was implemented in MATLAB (MATLAB ver. R2019a). The raw data and codes, along with explanations of all methods used for data analysis, have been published in a data repository [51]. All values were expressed as mean ± standard deviation, and no further statistical analysis was used.

Exfoliation reveals 10 nm diameter nanofibrils as fundamental building blocks
We employed ultrasonication, a purely mechanical method, to disintegrate T. clavipes silk in deionized water. Notably, this approach avoids harsh or hazardous chemicals that degrade the protein structure and compromise the biocompatibility of silk [25,52]. After 30 minutes of ultrasonication, the solution was cloudy, and optical microscopy revealed that the macroscopic fibers had begun exfoliating (Fig. 2, A and B). Scanning electron microscopy (SEM, Fig. 2D) of the exfoliated material further revealed the fibers' internal fibrillar structure. Toward the end of each fiber fragment (Fig. 2C), exfoliation had progressed all the way to the core, eventually compromising the integrity of the fiber as a whole. The process decomposed the fiber into thinner and thinner fibrils (Fig. 2F), successively decreasing diameters from 300 to 40 nm (Fig.   2, G and H). No particular fibril diameter was dominant, especially when volume averages were considered (Fig. 2H). The latter were obtained by weighting the fibril counts with the square of the fibril diameter, representing the cross-sectional area of the fibrils. This observation indicates the absence of nanofibril bundles of a particular diameter, contrary to what has been suggested in the literature [13,53,54].
To observe material that was completely removed from the fiber in this process and ended up in solution, we spin-coated a small droplet of the solution onto a mica substrate and imaged it using dynamic-mode AFM. Interestingly, the AFM images showed a homogeneous population of nanofibrils with diameters of ≈10 nm (Fig. 2E). The fact that we do not observe the smallest, 10 nm-diameter nanofibrils in our SEM images is most likely due to the resolution limitations of our hardware. Furthermore, images taken on dried samples may underestimate the degree of exfoliation (and perhaps overestimate the fibril diameters) due to the re-aggregation and re-assembly of already exfoliated fibrils in the drying process. Importantly, when we increased the ultrasonication duration to 135 minutes, virtually no fiber fragments were detectable in the solution using optical microscopy. AFM analysis after spin coating this solution onto mica (Fig. 3A) revealed nanofibrils similar to Fig. 2E. Therefore, we conclude that this longer ultrasonication fully exfoliated the 3.4 mg of silk into a solution of nanofibrils with a concentration of 0.09 mg/mL. Interestingly, analysis of the AFM images suggested that the elongated sonication time did not further disintegrate the nanofibrils themselves. Furthermore, AFM images that were taken after 135 min (Fig. 3A) showed a much smaller amount of background material between the fibrils compared to AFM images taken after 30 min of sonication (Fig. 2E), where a significant amount of this non-fibrillar background material was present. This may seem counterintuitive, because one might assume that longer sonication time has the ability to break the fibrils into smaller fragments, thus leading to more non-fibrillar background. Our observation, however, can be explained by the fact that a natural T. clavipes MA fiber features an outer glycoprotein coating [20,55], which easily dissolves in water. (Assuming a 200 nm-thick [20,55] glycoprotein layer and a 4 μm-diameter fiber, the glycoprotein accounts for ≈20 vol% of the fiber.) These dissolved glycoproteins thus represent a much larger fraction of the solution protein in the early exfoliation process (Fig. 2E). Sonication for longer times then reduces the relative amount of unorganized background protein, as more nanofibrils from the fiber are exfoliated into the solution (Fig. 3A).
In essence, our exfoliation experiments suggest that, except for the soluble glycoprotein surface layer, virtually the entire T. clavipes MA silk fiber consists of ≈10 nm-diameter fibrils that were separated using relatively mild mechanical techniques. Since the non-fibrillar material of fully exfoliated fibers can be attributed to the glycoprotein fiber coating, we can conclude that the nanofibrils are the overwhelmingly dominant structural elements of these silk fibers. We did not observe any significant hierarchical organization of nanofibrils between their 10 nm size and the macroscopic silk fiber, i.e. there were no nanofibril bundles of a preferred size within the fiber. Instead, our SEM images of disintegrated fibers show branches with a wide range of diameters (Fig. 2, C, F, and H), possibly disconnected from the main fiber at random locations.
The SEM evidence in Fig. 2C and 2D further shows that the separation of fiber components occurs strictly parallel to the fiber axis, suggesting that all fibrils are oriented parallel within the fiber. This is confirmed by AFM evidence of fiber fragments not completely exfoliated, where this parallel organization is revealed at the level of nanofibrils (inset in Fig. 3A). These observations are also in line with an NMR-based study conducted on Trichonephila edulis spider silk [54]. Based on these findings, we have developed the structural model of a T. clavipes MA fiber shown in Fig. 3B.
Notably, while the fiber completely disintegrated under ultrasound exposure, the nanofibrils themselves proved comparatively robust. This is in line with our recent study showing that silk of the Chilean recluse spider features highly anisotropic mechanical properties [56]. The work showed that the mechanical forces needed to separate recluse silk nanofibrils are five times smaller than the nanofibrils' tensile breaking forces. The work further suggested that van der Waals forces dominate interfibrillar binding. Corresponding conjectures regarding the internal mechanics of T. clavipes silk fibers are feasible. Facile ultrasound exfoliation of T. clavipes fibers would be in line with relatively weak van der Waals bonding between nanofibrils.

Spin-coating self-assembles native proteins into 10 nm nanofibrils
Having revealed that ≈10 nm-diameter fibrils, organized in a parallel fashion, are the fundamental building block of a T. clavipes MA fiber, the question of how these fibrils are formed and organized into a parallel bundle becomes very important, especially for the goal of making synthetic fibers inspired by spider silk. Building on some of our earlier observations on silkworm silk [38], we thus studied the process of nanofibril formation via self-assembly. We isolated major ampullate silk glands of T. clavipes spiders to extract their silk dope in native form as an aqueous gel. This gel was then diluted to 100 mg/L in deionized water and applied to freshly cleaved mica substrates via spin coating. This procedure reduces the Na + and Cl − concentrations, it reduces pH to ≈5.6 through adsorption of CO 2 followed by the production of carbonic acid, and it introduces shear. All of these conditions are known to also occur in the spider's spinneret at the onset of spinning and have been suggested to be triggers for spidroin self-assembly [47][48][49]. The obtained samples were imaged using dynamic-mode AFM to investigate the structural conformation of the protein. A typical AFM image is shown in Fig. 3C, revealing nanofibrils with a high degree of uniformity in terms of their diameters and heights.
Interestingly, regarding their diameter, shape, and length, these self-assembled nanofibrils were very similar to the nanofibrils obtained via exfoliation of a native silk fiber from the same spider species (Figs. 2E and 3A).

The correspondence of self-assembled and exfoliated structures underlines the spidroin's tendency to nanofibrillate
Using quantitative analysis of the fibril diameters, we found both exfoliated nanofibrils and self-assembled nanofibrils featured diameters of (10±2) nm (Fig. 3, D and E). Furthermore, the self-assembled nanofibrils tended to aggregate side by side along the axial direction, just as observed in the exfoliated material. Examples for such bundles of parallel nanofibrils are magnified in the insets of Fig. 3A and 3C. We believe that the striking similarity between the nanofibrils produced via exfoliation (Fig. 3A) and self-assembly (Fig. 3C), as well as their tendency to align in a parallel fashion, is not a coincidence. Rather, we are convinced that these nanofibrils were formed based on the same underlying process, following an inherent capability implemented in spidroin. In the natural spinning process of the fiber, the silk dope is moved downstream in the silk gland, which induces significant shear, followed by removal of water [57] and ion exchanges [47][48][49]. It has been suggested that shear is a critical trigger for the formation of nanofibrils [58,59]. Similarly, in our spin-coating process, the silk solution is exposed to shear as the centripetal forces accelerate it radially away from the substrate, a process that gradually increases the silk concentration and ultimately removes liquid water entirely. While both processes share the presence of shear and removal of water, several other parameters may be significantly different, such as time-vs-concentration and time-vs-shear rate profiles.
Nevertheless, the fact that they yield nanofibrils of almost identical morphologies suggests that the nanofibril formation mechanism in silk is relatively robust.

Spin-coating is applied to reveal the triggers of self-assembly
Based on the ability of our spin coating procedure to yield nanofibrils virtually identical to the natural spinning process, we can use this approach to learn more about the self-assembly of spidroin at the molecular scale. In a previous study, we found that the native silk protein of the silkworm Bombyx mori tends to open up and denature at low concentrations [39]. Therefore, we first explored the influence of protein concentration. We diluted the native protein concentration further, to 10 mg/L, using deionized water and spin-casted this solution onto mica sheets.
Interestingly, the protein did not form long, straight nanofibrils, as observed at higher concentrations (Fig. 3C). Instead, the spidroin formed relatively loose and open structures (Fig.   4A) under these conditions, typically covering an area of 50 nm or more in diameter. The curly strands featured apparent widths of only 5-6 nm, about half of what we observed in the fibrils self-assembled at higher concentrations, and apparent heights of ≈0.2 nm. The true width of these strands is most likely significantly less because AFM generally overestimates the width of objects substantially smaller than the tip. We suspected that these curly strands represent denatured spidroin molecules that unfolded under low-concentration conditions. To further corroborate this hypothesis, we first estimated the lengths of the backbones of the fully unfolded single molecule of major ampullate silk proteins (MaSp). The silk of T. clavipes has been shown to feature two major components, MaSp-1 and MaSp-2 with ≈747 and ≈3,146 amino acids, which correspond to lengths of ≈260 nm and ≈1100 nm, respectively [60,61], assuming a length of 0.35 nm per amino acid [62]. Surprisingly, tracing the lengths of several of the curly strands in Figure 4a, we found good agreement with these lengths, supporting our hypothesis, and in line with our previous findings for the silkworm's native silk proteins [38]. Moreover, the dimensions of these structures have a surprising resemblance to what Parent et al. proposed as the hydrodynamic radius (r H ) of the major ampullate silk monomer of the black widow spider (Latrodectus hesperus) based on NMR experiments [43]. Based on their measured r H ≈ 25 nm, they illustrated the spidroin monomer as shown in Fig. 4B [43]. Our results directly visualize the actual spatial conformation of the spidroin in the low-concentration regime for the first time, and the similarity of the images thus provides support for the model developed by Parent et al. We note that AFM revealed slightly larger aggregate sizes. This could be caused by the AFM sample preparation process, in which the proteins are forced into a 2-dimensional (2D) conformation on the substrate, potentially increasing their lateral spread in comparison to the true 3D conformation in solution probed by NMR. Importantly, our results showing that spidroin denatures at low concentrations mean that it may lose the ability to self-assemble into nanofibrils under these conditions, and thus reveal the spidroin concentration as another important factor in the self-assembly process. Hence, our experimental method can also identify the threshold concentration of silk proteins needed for self-assembly. Finally, we studied the influence of pH or concentration of Na + and Cl − ions on selfassembly, which have previously been suggested to be deciding factors in the spinning process [47][48][49]. Therefore, we diluted the native dope from the glands in PBS buffer at pH 7.4 rather than in deionized water. PBS buffer maintains a relatively high concentration of Na + and Cl − ions in the solution and keeps the pH neutral, akin to conditions in the storage sack of the spider.
Spin-coated AFM samples made from such solutions at higher concentrations of 1000 mg/L no longer showed long, straight protein nanofibrils. Instead, the protein was organized in islands with 200-300 nm in diameter (Fig. 4E). The inset reveals that these islands are clusters of short protein fibril segments with diameters of ≈10 nm (similar to what we observed in unbuffered solutions, as shown in Fig. 3C) and relatively short lengths, in the range of 50-90 nm. The area between the islands appears to be composed of globules.

Chemical and physical triggers during self-assembly
The above experiment suggests that shear alone is not sufficient to trigger the full transformation of spidroin into long nanofibrils; instead, application of shear leaves the protein in a pre-assembled state, where a fraction is present as short, elongated, pre-fibrillar assemblies, of L. hesperus spidroin (Fig. 4D) with respect to fibril diameters, fibril lengths, and size of assemblies. They diluted the samples in urea, which prevents the reduction of pH toward acidic conditions, and even though they applied shear to their solutions, they only observed transformation of the silk proteins from globular assemblies (Fig. 4C) into pre-fibrillar structures, but not complete assembly into long nanofibrils. Their observations are, thus, in close agreement with our observations and conclusionsthat shear alone is not sufficient to trigger nanofibrillar assembly.
Our observation that the reduction of pH and Na + and Cl − concentrations are needed as well to trigger nanofibrillar assembly is in line with previous studies focusing on the functions of spidroin's N-terminal domain during self-assembly. One study used computational modeling to show that a high concentration of sodium chloride would hamper the dimerization of Euprosthenops australis spidroin's N-terminal domain, a key step to fibrillation [46], while another study revealed that neutral pH would also impede this step to avoid aggregation by utilizing recombinant -mini-spidroins‖ [63]. What our findings demonstrate is that the natural system uses several independent factorsconcentration of Na + and Cl − , pH, and sheareach of which is necessary to trigger the formation of nanofibrils. In the storage sack, none of the conditions necessary for assembly are met, which provides an effective mechanism to prevent premature assembly of the spidroin.

Conclusion
We have shown that ≈10 nm-diameter nanofibrils play a decisive role in the MA silk of the golden silk orb-weaver T. clavipes. We were able to virtually completely decompose natural spider silk fibers into such nanofibrils for the first time, as well as synthesize nanofibrils with almost identical morphology through self-assembly from native silk dope. We interpret this to mean that silk protein has a strong intrinsic mechanism to form nanofibrils of a defined morphology. To trigger this strong fibril-forming mechanism, several independent physical and chemical conditionsshear, reduction of pH, Na + , and Cl − concentrationsneed to be met simultaneously. Here, we have demonstrated for the first time that the absence of any of these triggers prevents premature assembly of spidroin in the storage sack and thus provides conditions enabling long-term storage. Importantly, there is a strong correlation between AFM, NMR, and cryo-TEM experiments in terms of the observed protein structures across a range of different experimental parameters (Fig. 4). This remarkable agreement of results from complementary methods suggests that these experiments observe the correct structure and that the information obtained can be combined toward a consensus model.
Notably, the ≈10 nm-diameter nanofibrils were the only structural element we observed throughout all exfoliation stages after the initial glycoprotein layer had dissolved. This shows that the entire core of the fiber consists of such nanofibrils, and we showed that they are organized in a parallel fashion. Moreover, our exfoliation results did not show evidence of any systematic hierarchical organization among nanofibrils, i.e., we did not see nanofibril bundles of distinct diameters others had suggested [13,53,54]. We used our evidence to develop a refined structural model of T. clavipes spider silk fibers (Fig. 3B).
Our work has shown two complementary nanofibril preparation pathways, disassembly of micro-sized fibers (top-down) vs. self-assembly of molecules (bottom-up), to make spider silk in this form for the first time, with great potential for new applications [25][26][27]30]. In addition, the methods we introduced provide new ways to study the role of nanofibrils, their formation, and organization. A limitation of our method, the combination of spin coating with AFM, is that it does not fully replicate the natural spinning system of spiders. In the natural system, the viscous spidroin solution self-assembles into nanofibrils and ultimately transforms into a microscopic fiber. Although our in-vitro approach has the capability to study the parameters of nanofibril self-assembly, it is not yet developed to create microscopic fibers as the end product. However, our method unlocks a territory to study the most important stage of the fiber formation, which is still a missing link in the research of silk synthesis, i.e., the conditions of nanofibrils formation. Currently, most synthetic silks are inferior to their natural counterparts in terms of their mechanical properties [47,[64][65][66]. We believe that this is because the structure of the natural silk fiber, including the critically important nanofibrils, has not been fully reproduced, yet [65,66]. Our system provides a relatively easy-to-implement yet versatile in-vitro approach to evaluate the influence of different physical and chemical parameters on self-assembly. It can be used to optimize the spinning parameters of synthetic silks made from recombinant silk proteins to yield fibers structurally closer to natural silk. We believe our findings will have a significant impact on ongoing efforts to mass-produce desirable high-performance materials inspired by spider silk.


extfieldsofstudy:
Computer Science
Engineering

text:
Synthetic magnetic resonance images for domain adaptation: Application to fetal brain tissue segmentation

The quantitative assessment of the developing human brain in utero is crucial to fully understand neurodevelopment. Thus, automated multi-tissue fetal brain segmentation algorithms are being developed, which in turn require annotated data to be trained. However, the available annotated fetal brain datasets are limited in number and heterogeneity, hampering domain adaptation strategies for robust segmentation. In this context, we use FaBiAN, a Fetal Brain magnetic resonance Acquisition Numerical phantom, to simulate various realistic magnetic resonance images of the fetal brain along with its class labels. We demonstrate that these multiple synthetic annotated data, generated at no cost and further reconstructed using the target super-resolution technique, can be successfully used for domain adaptation of a deep learning method that segments seven brain tissues. Overall, the accuracy of the segmentation is significantly enhanced, especially in the cortical gray matter, the white matter, the cerebellum, the deep gray matter and the brain stem.

INTRODUCTION
Morphometric measurements in the developing fetal brain are critical as many congenital disorders are related to a disrupted growth of the structures [1]. Magnetic resonance imaging (MRI) is a powerful tool to investigate equivocal neurological patterns. However, images are often corrupted by random movements of the fetus which cause signal drops. Superresolution (SR) reconstruction techniques take advantage of * Hélène Lajous and Meritxell Bach Cuadra contributed equally to this work. the redundancy between orthogonal low-resolution (LR) series to reconstruct an isotropic high-resolution volume of the fetal brain with reduced intensity artifacts and motion sensitivity [2,3,4,5,6]. Subsequent multi-tissue segmentation is key for advanced quantitative analysis of the fetal brain [7,8,9,10,11]. Although manual annotation is a cumbersome and tedious task prone to human error, it is a prerequisite for the training of supervised deep learning approaches that in turn enable accurate automated delineation of fetal brain tissues [8,9,10,12].
Despite two recent public releases of annotated datasets [11,13] that support the development of accurate automatic deep learning segmentation methods, their generalization across different domains remains barely explored [14]. Specifically, the training of a deep learning network relies on an optimization problem to fit a data distribution, and tends to perform poorly on a new dataset with a different distribution due to variations of MR vendors, acquisition parameters or image reconstruction techniques. Therefore, while a segmentation framework can apply successfully to a specific dataset, it might fail on a different but related dataset. Domain adaptation addresses this data distribution gap by generalizing a model from one domain to another. It consists in using knowledge learned in a source domain to adapt a model to a target domain. Transfer learning has recently proven to be effective in adapting automatic segmentation methods for fetal brain MRI from different reconstruction methods [14].
In this paper, we explore different domains due to different SR reconstruction methods, as it often occurs when dealing with datasets from multiple hospitals and research teams. We thus study how synthetic MR images, which mimic our data target domain, can help to adapt a tissue segmentation network trained from another domain.

METHODOLOGY
The framework displayed in Fig. 1 provides an overview of the datasets (A and B) and the experimental design (C).

Domain gap
SR reconstruction techniques rely on several pre-processing steps (denoising, bias field correction, intensity normalization, etc.), and different direct model formulation and regularization approaches. This leads to SR reconstructed images that might be very different in terms of intensity and sharpness, as previously discussed in [14], and thus generate different domains. We define the following domains (see Fig. 2): Source domain SR obtained with a simplified version of the Image Registration Toolkit (SIMPLE IRTK) [3] under Licence from Ixico Ltd (FeTA-irtk), Target domain MIALSRTK pipeline [5,15] (FeTA-mial), Target-like domain Numerically-simulated images SRreconstructed with MIALSRTK (FaBiAN-mial).

Clinical datasets (FeTA-irtk and FeTA-mial)
Among the 80 subjects in the publicly available Fetal Tissue Annotation Dataset (FeTA 2.0) [11], 40 were reconstructed with an isotropic resolution of 0.8594mm (FeTA-irtk) and 40 with an isotropic resolution of 0.5mm (FeTA-mial). The SR images of this latter set was resampled to an isotropic resolution of 0.8mm and annotations were refined [16]. The extra-axial cerebrospinal fluid spaces (CSF), the cortical gray matter (GM), the white matter (WM), the ventricular system (lateral, third and fourth ventricles), the cerebellum, the deep gray matter (dGM) and the brain stem were manually annotated in the resulting FeTA-irtk and FeTA-mial SR volumes. An engineer with 20 years of experience in medical image processing assessed the quality of the 3D SR reconstructions.
Only subjects with excellent (good quality without any blurring) and acceptable (overall good quality with some blurring but still relevant for diagnosis purposes) SR were considered in the following. Thirty-one subjects (15 neurotypical and 16 pathological subjects in the gestational age (GA) range of 20.0 to 33.4 weeks) from FeTA-mial and 37 subjects (17 neurotypical and 20 pathological subjects in the GA range of 20.1 to 34.8 weeks) from FeTA-irtk were kept after quality control.

Simulated data in target-like domain (FaBiAN-mial)
The SR label maps from 17 clinical cases (nine neurotypical and eight pathological subjects in the GA range of 20.9 to 34.8 weeks) among the 37 FeTA-irtk subjects serve as fetal brain models to generate realistic synthetic images of the fetal brain throughout development. Typical T2-weighted singleshot fast spin echo (SS-FSE) acquisitions of the fetal brain are simulated at either 1.5 T or 3 T using FaBiAN, a Fetal Brain magnetic resonance Acquisition Numerical phantom [17,18]. For every subject, partially-overlapping series of 2D thick slices are simulated in each of the three orthogonal orientations with random little or moderate motion.
The simulated LR images are interpolated to 0.8594 × 0.8594mm 2 in the in-plane direction to match the resolution of the clinical SR reconstructions. Automated segmentation of the fetal brain is propagated in all LR image series from the initial SR label maps by a nearest-neighbour interpolation using FaBiAN simulation framework [18]. The fetal brain and its corresponding segmentation are SR-reconstructed using the docker version of the MIALSRTK pipeline [5,19], adapted to reconstruct associated label maps (FaBiAN-mial).

Segmentation networks
Configurations. The popular 2D U-Net [20] architecture is selected as it recently proved its ability to deal with fetal brain MRI tissue segmentation [8,14]. It is run in the different training configurations described below ( Fig. 1-panel C): • A Gold Gtantard (GS) network is trained using 20 randomly selected subjects from the FeTA-mial target domain (N=20, target domain). • A Baseline network is trained on the full FeTA-irtk set (N=37, source domain). • An Initializer (Init) network is trained on the 20 FeTA-irtk subjects that are not used for simulation purposes (N=20, source domain). • DA FaBiAN Baseline and DA FaBiAN Init are initialized respectively with Baseline and Init pre-trained weights to perform transfer learning using the 17 FaBiANmial subjects (N=17, target-like domain). A pure testing set that consists of the 11 remaining FeTAmial subjects (five neurotypical and six pathological subjects) is used to compare the models.
Strategy. The networks are trained on 64 × 64 voxel size image patches using a multi-view approach, considering intracranial voxels only. All patches are duplicated and the fol-lowing data augmentation is randomly applied: rotation, flipping and addition of Gaussian noise. Intensities of all patches are standardized. A cross-validation strategy is adopted to tune the learning rate decay. All networks are trained by optimizing a hybrid loss function that combines, with equal contribution, a categorical cross-entropy loss and a Dice similarity coefficient (DSC).
At test-time, overlapping patches are inferred from the three orientations and final prediction is reconstructed using a majority voting strategy.
Evaluation. The performance of the networks is evaluated with the DSC [21] between the ground truth manual annotations and the predicted segmentation. A paired Wilcoxon rank-sum test is performed between each experimental configuration (DA FaBiAN Init and DA FaBiAN Baseline) and the Baseline. For individual fetal brain tissues, p-values are adjusted for multiple comparisons using Bonferroni correction. Statistical significance level is set to 0.05.

RESULTS
Qualitative results are shown in Fig. 3 for a fetus of 31.2 weeks of GA. Table 1 shows the mean DSC ± standard deviation computed for every segmented fetal brain tissue in each configuration studied. Overall, the performance of the segmentation algorithm is significantly enhanced when finetuning the Baseline network with synthetic, yet realistic fetal brain MR images reconstructed in the target-like domain (DA FaBiAN). The accuracy of the segmentation algorithm is slightly decreased in the ventricles only, but without statistical significance. This may be due to erroneous label propagation (after simulation and reconstruction) resulting in neighboring areas of similar intensities at the mid-sagittal plane either segmented as CSF or ventricles. Conversely, the segmentation of the cerebellum is even more accurate when complementing the Baseline dataset with synthetic images (DA FaBiAN) than when training the network directly with the target FeTAmial dataset (Gold Standard).
Interestingly, our domain adaptation strategy leads to a more accurate segmentation of the CSF, the cortical GM, the WM, the cerebellum, the deep GM and the brain stem in pathological cases than the Baseline, as shown in Fig. 4. It is also the case for the ventricles when combining domain adaptation and data augmentation approaches (DA FaBiAN Baseline). The trend is different in neurotypical subjects, where DA FaBiAN Baseline only results in an improved segmentation of the deep GM, the brain stem and the cerebellum. The segmentation of the latter is even more accurate than when training the network on clinical data in the target domain (GS). However, the performance of the segmentation algorithm is stable for the CSF and the cortex, with a slight increase for the WM in the configuration DA FaBiAN Baseline, whereas it decreases for the ventricles.

CONCLUSION
We have demonstrated for the first time that synthetic numerically-generated MR images of the developing fetal brain can be used for transfer learning between two different domains. The proposed domain adaptation strategy has proven to be powerful since it is free of human intervention in the sense that it does not require expert annotations in the target domain. Thus, it overcomes the lack of clinical annotations and can be generated from any pre-existing label map. Overall, our method significantly enhances the performance of the baseline fetal brain tissue segmentation algorithm. Future work will further extend the idea of domain adaptation to multi-scanner multi-center datasets.